Lipases (triacylglycerol hydrolase E.C. 3.1.1.3) are defined as hydrolytic enzymes that act on the ester linkage in triacylglycerol in aqueous systems to yield free fatty acids, partial glycerides and glycerol. This group of enzymes under low water activity is capable of catalyzing their reverse hydrolysis reaction. The reverse catalytic activity of lipases has been widely exploited for the synthesis of valuable compounds that contain ester and amide linkages or other related chemicals containing functional groups such as hydroxyl, carboxylic and amino groups. In particularly, lipases have been utilized for reforming fats, oils, waxes, phospholipids and sphingolipids to obtain new desired functional properties, and for separating optically active compounds from their racemic mixtures. Of particular interest, the use of a multi-enzyme system comprised of different lipases immobilized on a polymeric support will be disclosed for the synthesis of fatty acid short-chain alkyl esters (biodiesel).
Currently, there are more than 40 different lipases and phospholipases commercially available however only a few of them are prepared in commercial quantities. Some of the most industrially promising interfacial enzymes are derived from Candida antarctica, Candida rugosa, Rhizomucor miehei, Pseudomonas sp., Rhizopus niveus, Mucor javanicus, Rhizopus oryzae, Aspergillus niger, Penicillium camembertii, Alcaligenes sp., Burkholderia sp., Thermomyces lanuginosa, Chromobacterium viscosum, papaya seeds, and pancreatin.
The most familiar enzyme immobilization techniques are in general divided according to the following:                1. Physical adsorption of enzymes to solid supports, such as silica and insoluble polymers.        2. Adsorption on ion-exchange resins.        3. Covalent binding of enzymes to a solid support material, such as epoxidated inorganic or polymer supports.        4. Entrapment of enzymes in a growing polymer.        5. Confinement of enzymes in a membrane reactor or in semi-permeable gels.        6. Cross-linking enzyme crystals (CLECS's) or aggregates (CLEAS's).        
Physical adsorption of lipases based on use of polymeric supports with high porosity or use of ion-exchange resins are the most practiced immobilization methods for lipases. This method is characterized with its simplicity and yielding reliable synthetic activity.
The use of free or immobilized lipases for transesterification of triglycerides and short-chain alcohols to form fatty acid alkyl esters has yielded unsatisfactory results with respect to activity and stability of the enzyme. Also, the cost-effectiveness of the immobilized enzymes, for carrying out enzymatic production of fatty acid alkyl esters at industrial quantities, is still prohibited. Furthermore, it has been reported that all currently available lipases in either their free or immobilized forms are incapable of reaching near to complete conversions, preferably above 99%, for oil triglycerides to fatty acid alkyl esters at reasonable reaction time, particularly below 8 hours.
Another major drawback of lipases results from their low tolerance towards hydrophilic substrates, particularly short-chain alcohols, short-chain fatty acids (both below C4), water and glycerol typically present in the transesterification reaction medium. It has been observed in many research studies that short-chain alcohols and short-chain fatty acids, such as methanol and acetic acid, respectively, are responsible for detaching essential water molecules from the quaternary structure of those enzymes, leading to their denaturation and consequently loss of their catalytic activity. Also, the presence of such hydrophilic molecules in the reaction medium, results in detaching the enzyme molecules from the support and consequently decrease in the enzyme operational life-time. Therefore, it is not surprising that the application of lipases for production of commercial quantities of fatty acids methyl esters “biodiesel” using oil triglycerides and methanol as substrates is infeasible.
Use of mixtures of lipases has been suggested [Lee, D. H. et al., Biotechnology and Bioprocess Engineering 2006, 11:522-525]. This publication describes production of bio-diesel using a mixture of chemically bound, immobilized Rhizopus oryzae and Candida rugosa lipases. As can be seen, the reaction time was relatively long, typically more than 24 hours to reach conversions over 96% to biodiesel. Also, the results presented in this publication show that the mixture of enzymes used lost more than 20% of its initial activity after as few as 10 cycles of use. This may be attributed to the accumulation of partial glycerides intermediates in the reaction system, which decrease the transesterification reaction and thus prolong the reaction time. The deactivation of the biocatalyst in the system described in this publication is a key drawback, which prevents its industrial application.
It is therefore an object of this invention to provide a new method for obtaining highly active and stable immobilized lipases for the synthesis of fatty acids alkyl esters, especially fatty acid methyl esters for use as “biodiesel”.
It is a further object of the present invention to provide highly active, and stable, immobilized multi-enzyme preparation which possesses high tolerance towards short-chain alcohols and short-chain fatty acids, especially methanol, ethanol and acetic acid respectively, and other polyols such as glycerol, as well as other inhibiting factors typically present in oils and fats, in particularly of inedible grade.
It is a further object of the present invention to provide a one-step or multi-step enzyme reactor configuration for obtaining the desired product, namely, fatty acid alkyl esters at near to complete conversions during reasonable reaction time, typically below 5 hours.